BICD 110 Study Guide - Spring 2018, Comprehensive Midterm Notes - Protein, Cytoplasm, Endoplasmic Reticulum
BICD 110
MIDTERM EXAM
STUDY GUIDE
Fall 2018
BICD110 Lecture 1 Notes 4/3/18
- Microscopy
oCell is below limit of resolution of the human eye. We use microscopy to visualize cells
Average animal cell is 10-20 µm in diameter
The human eye can see a 100 µm object
obasic format of light microscope
- 2 factors important in whether an object can be seen
o1) magnification
o2) resolution
limit of resolution is the minimum distance 2 objects can approach another and still
appear separate
Limit of resolution for light microscope is ½ the wavelength of light. For visible light,
wavelength = 0.4 – 0.7 µm. Half wavelength = 0.2 – 0.35 µm, so objects bigger than
this will affect light and be visible, and objects small will not be visible because no
perturbation of waves will occur
Smallest objects visible with a light microscope are mitochondria and small bacteria,
which is 400x better than the naked eye
ousing a lens, objects can be magnified almost indefinitely
- problems of microscopy
o1) cells are 70% H2O, so not much detail can be seen
solution: use stains and dyes for different components of the cell, like DNA, proteins,
membranes, etc. These stains and dyes can bind to the components and refract light
o2) cells are fragile. They become distorted or broken by observation
solution: cells are usually fixed before observation to become stronger
2 types of fixations
methanol fixation: causes proteins to denature, precipitate out in place, and
complex to each other
chemical crosslinkers: formaldehyde or
glutaraldehyde used; interact with lysines in
proteins to complex together, allowing protein stabilization
o3) tissues are thick and light can’t penetrate
find more resources at oneclass.com
find more resources at oneclass.com
solution: embed tissue in something stronger than the tissue (like paraffin wax),
then slice into sections with microtome to allow 1-10 µm thick slices of tissue
o4) cells are killed by staining, fixing, and sectioning. There’s a possibility of looking at an
artifact (artificial fact because the cells are dead and look different)
solution: confirm results by looking at living cells
interference microscopy (phase contrast microscopy): used to
look at living cells with light instead of
staining/dying/fixing. As light passes from less
dense to more dense material, its speed slows.
The change in speed is used to enhance contrast
in living unstained cells
- Electron microscopy: used to observe small objects
smaller than 0.2 – 0.35 µm. When electrons are accelerated, they have
a characteristic wavelength. Electrons through 50,000 V acceleration
have wavelength of 0.005 nm
oLimit of resolution should be ½ wavelength = 0.0025 nm. However,
due to technical problems, the limit of resolution is 0.1 nm (still
400x better than light microscopy)
oStandard transmission EM (TEM): 50,000 – 100,000 V; 2D
Under right circumstances, can see DNA, ribosomes, and
proteins. However, same problems require fixing, embedding,
sectioning, and staining
Fix -> embed in plastic (very hard) -> section with diamond
knife (50 – 100 nm sections) -> stain with heavy metal solution (lead, uranium,
osmium) to make the objects they bind to dense
Can also stain with spray of heavy metal, called negative staining
oScanning EM (SEM): 3D
Put sample on grid -> coat with platinum -> use scanning EM machine to detect
Electron gun shoots electrons at coated sample. Detector moves around
sample and collects 2° electrons that have deflected off the platinum coat,
integrates them, and processed them into 3D picture on screen
Can magnify 20,000x. Good for fly eyes, bristles, pollen, and stereocilia on
cells in your ears
oHigh resolution scanning EM (FEISEM): 3D
Can magnify 100,000x. Good for seeing single microtubule or nuclear pore
Rare and expensive machines ($300,000+)
oFreeze fracture EM: 3D
freeze cell in liquid nitrogen (-196°C) -> fracture with knife -> coat
with metal -> view
Line of fracture is where the least amount of resistance occurs, like
in middle of membrane to expose proteins
oFreeze etch EM: 3D with better resolution
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Microscopy: cell is below limit of resolution of the human eye. Average animal cell is 10-20 m in diameter. The human eye can see a 100 m object: basic format of light microscope. 2 factors important in whether an object can be seen: 1) magnification, 2) resolution limit of resolution is the minimum distance 2 objects can approach another and still appear separate. Limit of resolution for light microscope is the wavelength of light. For visible light, wavelength = 0. 4 0. 7 m. Half wavelength = 0. 2 0. 35 m, so objects bigger than this will affect light and be visible, and objects small will not be visible because no perturbation of waves will occur. They become distorted or broken by observation solution: cells are usually fixed before observation to become stronger. Electron microscopy: used to observe small objects smaller than 0. 2 0. 35 m. However, due to technical problems, the limit of resolution is 0. 1 nm (still.
